All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Radiolabeled proteins, particularly antibodies, have been undergoing evaluation for many years as potential diagnostic and therapeutic reagents. Such reagents are thought to be particularly useful as cancer therapeutics, now that researchers are beginning to identify tumor-specific antigens and cognate ligands or antibodies which bind to such antigens. By administering a radiolabeled ligand or antibody which has binding specificity for a tumor-specific antigen, coupled to a radioisotope that has a short range, high energy and abundant particle emission, one has the potential to deliver a lethal dose of radiation directly to the tumor cell.
Depending on the particle range of the particular isotope, labels may be chosen based on their suitability for targeting a particular type of cell. For instance, gamma emitters are generally used for diagnostic purposes, i.e., visualizing tumors, but are generally ineffective as killing agents. In contrast, alpha and beta emitters may be used to effect cell killing. Alpha emitters may be particularly useful for blood-born diseases or vascular tumors where they can achieve good penetration; although one particle emission in some cases may be enough to effect cell killing, typically alpha emitter must be located right at the cell surface. In contrast, beta emitters, i.e., 90Y, are particularly suitable for bulkier, more localized disease because they typically have a longer emission range.
Yttrium-90-labeled antibodies and peptides in particular have shown encouraging results in clinical therapy protocols (Thomas et al. 1995. Gamma-interferon administration after 90Y radiolabeled antibody therapy: survival and hematopoietic toxicity studies. Int. J. Radiat. Oncol. Biol. Phys. 31: 529–534; DeNardo et al. 1995. Yttrium-90/Indium-111 DOTA peptide chimeric L6: pharmacokinetics, dosimetry and initial therapeutic studies in patients with breast cancer. J. Nucl. Med. 36: 97P). Such conjugates are usually made by coupling a bifunctional chelator to the protein or antibody, then conjugating the radiolabel to the protein construct via the bifunctional chelator. For instance, copending application Ser. Nos. 08/475,813, 08/475,815 and 08/478,967, herein incorporated by reference, describe radiolabeled therapeutic antibodies for the targeting and destruction of B cell lymphomas and tumor cells. Particularly disclosed is the Y2B8 conjugate, which comprises an anti-human CD20 murine monoclonal antibody, 2B8, attached to 90Y via a bifunctional chelator, MX-DTPA.
Patents relating to chelators and chelator conjugates are known in the art. For instance, U.S. Pat. No. 4,831,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelators and protein conjugates containing the same, and methods for their preparation. U.S. Pat. Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 of Gansow also relate to polysubstituted DTPA chelators. As described in Kozak et al., several DTPA chelating agents, including MX-DTPA, have been shown to be suitable for yttrium-monoclonal antibody radioimmunotherapy (1989. Nature of the bifunctional chelating agent used for radioimmunotherapy with yttrium-90 monoclonal antibodies: Critical factors in determining in vivo survival and organ toxicity. Cancer Res. 49: 2639–2644). These references are incorporated herein in their entirety.
Yttrium-90 is particularly suited for radioimmunotherapy and radioligand therapy for several reasons. The 64 hour half-life of 90Y is long enough to allow antibody accumulation by the tumor and, unlike e.g., 131I, it is a pure beta emitter of high energy (E max 2.27 MeV) with no accompanying gamma irradiation in its decay. It's particle emission range is 100 to 1000 cell diameters, which is a sufficiently minimal amount of penetrating radiation that outpatient administration would be possible. Furthermore, internalization of labeled antibodies is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells which might lack the target antigen.
However, despite the recognized utility of yttrium-labeled antibodies and the encouraging clinical results with some yttrium-labeled therapeutics, many patients are deprived of the benefit these therapeutics might offer because of the inherent difficulties in conducting both the radiolabeling and administration at a single location. This significant problem is evident in the nearly complete void of kits and products which enable on-site labeling of reagents with alpha and beta emitting radioisotopes, which might otherwise demonstrate the commercial applicability of such technology.
The problem with providing kits for radiolabeling and subsequent administration of therapeutics labeled with destructive isotopes appears to be the long-existing belief in the art that, before such therapeutics could be administered to a patient, an extensive purification process was required to remove unbound label so as not to expose the patient to free radioisotope which might accumulate in the bone and other non-target organs. Even those kits currently available for labeling antibodies with yttrium require a complicated purification step before the therapeutic is ready for administration.
For instance, Antisoma currently offers a kit for radiolabeling monoclonal antibody HMFG1 (Theragyn®) with 90Y for subsequent administration to patients who have been diagnosed with ovarian cancer. An extended phase I-II study demonstrated that this treatment may be particularly beneficial to patients as a follow-up to conventional surgery and chemotherapy (Hird et al. 1993. Adjuvant therapy of ovarian cancer with radioactive monoclonal antibody. Br. J. Cancer 68: 403–406). Yet Antisoma's labeling method requires removal of unbound label by Sephadex G50 gel filtration, which is a significant deterrent to the Theragyn® labeling kit achieving commercial success, as well as an obstacle for ensuring that this therapy is readily available for all ovarian cancer patients for whom it might serve to benefit.
The fact that such reagents currently require purification before administration has been and will continue to be a major deterrent in their availability to all patients who could benefit from such technology unless a simplified method is presented that allows physicians to quickly, efficiently and safely administer such reagents. For instance, a doctor in an outpatient setting does not have the time or facilities to purify a reagent by HPLC or gel filtration chromatography before administering the reagent to his patient. This means that additional facilities must be available on site for concurrent production of the reagent and immediate delivery to the doctor, which drastically increases the cost of the therapy and in some cases might require a patient to travel a significant distance to receive the therapy. Alternatively, the drug could be labeled off-site, which would require prior preparation and at least a short-term storage of the therapeutic. This not only has the effect of decreasing the strength of the radioisotope through radioactive decay during storage, but also leads to significant radiolytic damage to the structural integrity of the protein by overexposure to the radioisotope.
For instance, many reports have discussed the radiolytic nature of 90Y and similar radioisotopes (i.e., Salako et al. 1998. Effects of radiolysis on yttrium-90-labeled Lym-1 antibody preparations. J. Nucl. Med. 39: 667–670; Chakrabarti et al. 1996. Prevention of radiolysis of monoclonal antibody during labeling. J. Nucl. Med. 37: 1384–1388). As noted in Chakrabarti et al., radionuclides such as 90Y deliver a large amount of radiation to the antibody during the labeling process as well as during storage. Radiation has reportedly led to instances of significant antibody damage, which can eliminate preferential targeting of tumor cells and expose non-target tissues to significant levels of toxicity.
The mechanism for radiation damage has been attributed to the generation of free radicals (Pizzarello. 1975. Direct and indirect action. In: Pizzarello and Witcofski, eds. Basic Radiation Biology, 2nd ed. Philadelphia: Lea & Febger, pp. 20–29). But as noted in Salako et al., at an energy of 2.2 MeV, the beta particles emitted from 90Y could easily break most chemical bonds including the disulfide bridges of an antibody, which have a bond strength of only 4.4 eV (Skoog. 1985. Principles of Instrumental Analysis, 3d edition. San Francisco: Saunders). Thus, the shorter the amount of time that the protein to be labeled is exposed to destructive radioisotopes such as 90Y, the better the chances will be that the protein will maintain the structural integrity and binding specificity it requires to interact with the target antigen up until the time it is administered and reaches the target site.
The radiolytic nature of 90Y has been known in the art for years and many have tried to solve the problem 90Y presents in the commercial application of these therapeutics. For instance, both Salako et al. and Chakrabarti et al. evaluate the use of radioprotectants in 90Y-labeled antibody preparations as a means to decrease damage to the antibody. Salako et al. in particular reported that human serum albumin enabled maintenance of 90Y-labeled antibody immunoreactivity for up to 72 hours. However, the specific activity exhibited by Salako's preparations was rather low (less than 2 mCi/ml). Moreover, neither Salako nor Chakrabarti report any effort to forego the extensive purification processes required after antibody labeling. Salako et al. labels for a period of 45 minutes to an hour, then purifies the antibody by molecular sieve chromatography, whereas Chakrabarti labels for nearly three hours and purifies by gel filtration chromatography. Neither of these methods will be instrumental in bringing 90Y-labeled therapeutics to the out-patient setting.
Chinol and Hnatowich were able to achieve 90% radiochemical purity for 90Y-labeled proteins with specific activities ranging from 1–3 mCi/mg absent post-labeling purification, using their own generator-produced 90Y (1987. Generator-produced yttrium-90 for radioimmunotherapy. J. Nucl. Med. 28(9): 1465–1470). However, the authors expressly discourage administering preparations having less than 95% purity to patients, and suggest that HPLC may be an important and “possibly essential” step.
Those who have recognized that HPLC and other types of purification must be eliminated in the outpatient and hospital setting have not succeeded in developing a sufficient labeling protocol for 90Y such that a high level of label incorporation is achieved and an acceptable level of antibody stability is maintained. If a high level of radioincorporation is not consistently achieved, the patient could be exposed to unacceptable levels of free non-bound radiolabel if this label is not purified away from the reagent. Moreover, again, if antibody structural integrity is damaged such that the antibody loses target specificity, such reagents will not bind specifically to their cognate ligands.
Mather and colleagues set out with the purpose of labeling tumor-specific antibodies with 90Y in a manner such that post-labeling purification could be avoided (1989. Labeling monoclonal antibodies with yttrium-90. Eur. J. Nucl. Med. 15: 307–312). However, Mather found that high labeling efficiencies (over 95%) could only be achieved at modest specific activities (1 mCi/mg). Moreover, Mather et al. reports that their antibody preparations showed signs of breakdown (due to radiolysis) after only a few hours. This may be because Mather et al., as do many others in the field, conducted their labeling reaction over a period of one hour.
For example, there have been methods proposed for labeling protein reagents with less destructive labels such as 111In which forego additional purification steps. Richardson et al. propose such a procedure for labeling antibodies with 111In with the goal of facilitating a kit format for diagnostic use (Richardson et al. 1987. Optimization and batch production of DTPA-labeled antibody kits for routine use in 111In immunoscintography. Nucl. Med. Comm. 8: 347–356). However, the labeling method proposed in Richardson et al. is conducted over a period of one hour, which might be feasible with 111In which is not very radiolytic, but does not appear to be amenable to 90Y labeling applications as evidenced by the difficulties reported in Mather et al.
This brings us to the surprising and unexpected advantages of the present invention, which provides invaluable insight into the process of radiolabeling proteins with 90Y which has not been yet been recognized by others in the art. Surprisingly, the present inventors have found that the processes of HPLC or other purification steps that others have long thought to be necessary to achieve pure reagent, and the lengthy incubation times which others have adopted in an effort to increase the specific activity of their reagents, are actually detrimental to the process of preparing 90Y-labeled reagents. Such time-inclusive processes serve only to increase the damage to the protein due to radiolysis, leading to less specificity or binding, loss of radioisotope from the targeting agent and an increased rate of protein degradation by the time the radiolabeled protein is ready for injection. Surprisingly, the present inventors have found that efficient labeling with 90Y (>95% incorporation and at least 15 mCi/mg specific activity) can be accomplished in as little as two to five minutes, and in fact such labeling loses its efficiency as reaction times are increased beyond even eight minutes.
The fact that labeling with 90Y may now be achieved by the methods of the present invention in as little as one-two minutes or even as quickly as 30 seconds will completely dissolve the current skepticism in the field toward the applicability of yttrium radiolabeling kits in hospital and outpatient settings. The kits of the present invention will therefor finally satisfy the long felt need that has perhaps been recognized by many cancer patients and doctors alike with regard to the commercial applicability and accessability of protein-based, radiolabeled cancer therapeutics.